3,5-di(undecan-2-yl)heteropolycycle

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

, but deviation was related to source of S9 mix and did not influence the quality or integrity of the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Reference

Name:

Unnamed

Type:

Constituent

Method

Target gene:

induction of chromosome breakage

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix (phenobarbital and β-naphthoflavone induced)

Test concentrations with justification for top dose:

Pre-Experiment: 0.020; 0.039; 0.078; 0.16; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0 mM
Experiment I: without metabolic activation: 0.1; 5.0; 10.0 mM
Experiment I: with metabolic activation: 0.316; 1.0; 5.0; 10.0 mM
Experiment II: without metabolic activation: 0.1; 5.0; 10.0 mM
Experiment II: with metabolic activation: 0.4; 8.0; 10.0 mM

Vehicle / solvent:

The test item was dissolved in ethanol at a concentration of 1000 mM and was diluted in cell culture medium (MEM)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

V79 cells in vitro were widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells were chosen because of their relatively small number of chromosomes (diploid number, 2n = 22), their high proliferation rate (doubling time of the BSL BIOSERVICE V79 done in stock cultures: 12 - 14 h) and the high plating efficiency of untreated cells (normally more than 50%). These facts were necessary for the appropriate performance of the study.
The V79 (ATCC, CCL-93) cells are stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking for mycoplasma infections was carried out before freezing.
For the experiment, thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10E5 cells per flask were seeded in 15 mL o f MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3 - 4 days.

Evaluation criteria:

ACCEPTABILITY
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
- the number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0% - 4.0% (with and
without metabolic activation),
- the positive control substance should produce biologically relevant increases in the number of cells with structural chromosome aberrations.

EVALUATION
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells
(with and without metabolic activation)).

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results and a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data, both biological and thoroughly evaluated statistical significance should be considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

In experiment I and II with and without metabolic activation, no biologically relevant decrease of the relative mitotic index as indication of cell toxicity was noted at all concentrations evaluated. Furthermore, the cell density was not decreased below 70%.

In experiment I without metabolic activation the aberration rates of the negative control (l.5%)‚ solvent control (4.0%) and all dose groups treated with the test item [2.3% (0.1 mM)‚ 3.0% (5.0 mM) and 2.0% (10.0 mM)] were within the historical control data of the testing facility (0.0% - 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding solvent control. With metabolic activation, the aberration rates of the negative control (2.0%), solvent control (2.0%) and the higher dose groups treated with the test item [3.8% (1.0 mM), 2.0% (5.0 mM) and 3.0% (10.0 mM)] were within the historical control data of the testing facility (0.0% - 4.0%). An increase of the aberration rate above the historical negative control data of the testing facility was observed at a concentration of 0.316 mM (4.5%). A dose-response relationship was not found.

In experiment II without metabolic activation the aherration rates of the negative control (2.5%), solvent control (0.0%) and all dose goups treated with the test item [2.5% (0.1 mM), 3.0% (50 mM) and 0.5% (10.0 mM)] were within the historical control data of the testing facility (0.0% - 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding solvent control. With metabolic activation the aberration rates of the negative control (3.0%), solvent control (2.0%) and the highest dose group treated with the test item (l.5% (10.0 mM)) were within the historical control data of the testing facility (0.0% - 4.0%). The aberration rates found at the concentrations of 0.4 mM (6.8%) and 8.0 mM (5.0%) were clearly increased above the historical negative control data of the testing facility. Thus, the numher of aberrant cells found at these concentrations showed a biologically relevant increase compared to the corresponding solvent control without dose-response relationship.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item Sa 190 induced structural chromosomal aberrations in the V79 Chinese hamster cell line in presence of metabolic activation with S9 homogenate.
Therefore, the test item Sa 190 is considered to be clastogenic in this chromosome aberration test.

Executive summary:

To investigate the potential of Sa 190 to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out. The chromosomes were prepared 20 h after start of treatment with the test item, The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 0.1, 5.0 and 10.0 mM

with metabolic activation: 0.316, 1.0, 5.0 and 10.0 mM

Experiment II:

without metabolic activation: 0.1, 5.0 and 10.0 mM

with metabolic activation: 0.4, 8,0 and 10.0 mM

Precipitation of the test item was observed at the end of the treatment by the unaided eye in the experiment I with and without metabolic activation at concentrations of 5.0 mM and higher. In experiment II, precipitation of the test item was seen by the unaided eye without metabolic activation at concentrations of 5.0 mM and higher, with metabolic activation at concentrations of 8.0 mM and higher.

No toxic effects of the test item were noted with and without metabolic activation at all concentrations evaluated in experiment I and II In both experiments without metabolic activation no biologically relevant increase of the aberration rates was noted after treatment with the test item. With metabolic activation an increase of aberrant cells was found in experiment I at a concentration of 0.316 mM and in experiment II at concentrations of 0.4 mM and 8.0 mM

In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls. EMS ( 400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.